This project explores the molecular mechanism of active transport of neuro transmitters. In synaptic transmission two transport processes play a central role: high affinity, Na+-dependent transporters located in the plasma membrane and neurotransmitter-H+ antiporters located in synaptic vesicles. We have identified, purified and cloned prototypes of each class: GAT1, a sodium and chloride coupled gamma-amino butyric acid (GABA) transporter and VMAT-1, a vesicular monoamine-H+ antiporter. We will progress towards understanding the molecular mechanism by a) analysis of site-directed mutants with altered transport properties as well as of second site revertants, b) Study of the role of the loops connecting transmembrane alpha-helixes, c) determination of the topology and d) overexpression and 2D and/or 3D crystallization of the transporters. This approach is directed to understanding the structural basis of the function of both transporters. It is aimed to identifying specific residues and domains which play roles both in structure and function. This will deepen our insights not only into the mechanisms of neurotransmitter transport but also into those of ion-coupled solute transport in general. Since both GAT1 and VMAT1 function to regulate neurotransmitter levels at the synapse, understanding of their structures and function may provide important clues for their role not only under normal physiological conditions but also in disease. In addition, our studies will provide a more rational framework for the design of drugs for neurological disease.